WO2018132755A1 - Compositions et procédés pour perturber les mécanismes moléculaires associés à un dysfonctionnement mitochondrial et à une maladie neurodégénérative - Google Patents

Compositions et procédés pour perturber les mécanismes moléculaires associés à un dysfonctionnement mitochondrial et à une maladie neurodégénérative Download PDF

Info

Publication number
WO2018132755A1
WO2018132755A1 PCT/US2018/013645 US2018013645W WO2018132755A1 WO 2018132755 A1 WO2018132755 A1 WO 2018132755A1 US 2018013645 W US2018013645 W US 2018013645W WO 2018132755 A1 WO2018132755 A1 WO 2018132755A1
Authority
WO
WIPO (PCT)
Prior art keywords
alu
tomm
timm
modification
rna
Prior art date
Application number
PCT/US2018/013645
Other languages
English (en)
Inventor
Peter Anthony LARSEN
Original Assignee
Duke University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Duke University filed Critical Duke University
Priority to US16/477,645 priority Critical patent/US11352625B2/en
Publication of WO2018132755A1 publication Critical patent/WO2018132755A1/fr
Priority to US17/723,555 priority patent/US11932855B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the presently disclosed subject matter is directed to compositions and methods of disrupting the molecular mechanisms associated with mitochondrial dysfunction that contribute to disease, including neurodegenerative disease.
  • a hallmark of human neurodegenerative disorders is the accumulation of protein aggregates (e.g., fibrils, tangles, amyloid plaques, Lewy bodies, inclusion bodies) in the brain.
  • protein aggregates e.g., fibrils, tangles, amyloid plaques, Lewy bodies, inclusion bodies
  • AD Alzheimer's Disease
  • alpha- synuclein molecules i.e. Lewy Bodies
  • ALS amyotrophic lateral sclerosis
  • CTE chronic traumatic encephalopathy
  • Huntington's disease Mitochondrial dysfunction is hypothesized to play a role in the initial intra-cellular formation of these neuronal protein aggregates and subsequent inflammation and neuronal damage associated with multiple neurological disorders.
  • DNA-RNA and RNA-RNA hybridization has been utilized in a range of technologies including for nucleic acid detection and for alteration of gene expression.
  • Antisense nucleotides for example, disrupt gene expression by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, translation, and replication.
  • Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H, an activity that is present in most cell types.
  • Antisense molecules can be delivered into cells, as is the case for oligodeoxynucleotides (ODNs).
  • ODNs oligodeoxynucleotides
  • VITRAVENE for treatment of cytomegalovirus retinitis
  • VITRAVENE for treatment of cytomegalovirus retinitis
  • the present invention provides such compositions and methods for the treatment of neurodegenerative disease and other diseases and disorders resulting from dysfunctional mitochondrial populations.
  • the presently disclosed subject matter is directed to a method of modulating the function, expression, or both of a TOMM, TIMM, or APOE isoform polynucleotide in the cells of a subject.
  • the presently disclosed subject matter is directed to a method of preventing or treating a disease or disorder associated with at least one TOMM, TIMM, or APOE isoform.
  • the disclosed method comprises administering to a subject a therapeutically effective dose of a composition comprising at least one antisense oligonucleotide that binds to a TOMM, TIMM, or APOE isoform polynucleotide, thereby preventing or treating the disease or disorder.
  • the disease or disorder is a neurological disorder, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or Parkinson's disease.
  • the antisense oligonucleotide is an antisense RNA molecule, an antisense DNA molecule, an interference RNA (RNAi), a micro RNA, a decoy RNA molecule, a siRNA, an enzymatic RNA, or a therapeutic editing RNA.
  • RNAi interference RNA
  • the antisense oligonucleotide has at least 50%, 75%, or 95% sequence identity to a reverse complement of the TOMM, TIMM, or APOE isoform polynucleotide.
  • the isoform is an Alu element-induced isoform, such as an Alu element-induced TOMM40 isoform.
  • the TOMM40 isoform comprises the target region set forth in SEQ ID NO: 119.
  • the antisense oligonucleotide is selected from SEQ ID NOs: l-ll.
  • the composition comprising the antisense oligonucleotide is administered intraventricularly, intranasally, intrathecally, or systemically to the subject.
  • the subject is a human.
  • the presently disclosed subject matter is directed to a composition comprising one or more antisense oligonucleotides, wherein the antisense oligonucleotide has at least 50%, 75%, or 95% sequence identity to a reverse complement of a TOMM, TIMM, or APOE isoform polynucleotide.
  • the TOMM, TIMM, or APOE isoform polynucleotide is an Alu element-induced isoform, such as a TOMM40 isoform comprising the target region set forth in SEQ ID NO: 119.
  • the antisense oligonucleotide is selected from SEQ ID NOs: l-ll.
  • the antisense oligonucleotide can comprise one or a combination of chemical modifications comprising phosphate backbone modifications, phosphorothioate (PS) backbone modification, ribose sugar group modifications, 2'-0-methyl (20Me) modification, 2'-0-methoxy-ethyl (MOE) modification, locked nucleic acid (LNA) modification, tricyclo-DNA (tc-DNA) modification, 2'-fluoro modification, S -constrained-ethyl (cEt) modification, peptide nucleic acid (PNA) modification, or phosphorodiamidate morpholino oligomer (PMO) modification.
  • the composition can further comprise a pharmaceutically acceptable carrier.
  • the presently disclosed subject matter is directed to a method of reducing or eliminating the risk of neurodegenerative disease in a subject.
  • the method comprises modifying the Alu elements, regions immediately flanking the Alu elements, or both within one or more genes of the TOMM complex, the TIMM complex, or APOE.
  • the modifying comprises contacting the cells with one or more epigenetic regulator, such as DNA epigenetic activators, DNA epigenetic repressors, histone epigenetic activators, histone epigenetic repressors, or combinations thereof.
  • the modifying comprises targeted DNA mutation, targeted DNA excision, or combinations thereof.
  • the subject is a human.
  • the presently disclosed subject matter is directed to a method of modulating the function, expression, or both of a TOMM, TEVIM, or APOE isoform polynucleotide in the cells of a subject.
  • the method comprises contacting the cells with an antisense oligonucleotide, wherein the oligonucleotide has at least 50%, 75%, or 95% sequence identity to a reverse complement of the TOMM, TEVIM, or APOE isoform polynucleotide, thereby modulating function, expression, or both of the isoform.
  • the modulating occurs in vivo or in vitro.
  • the antisense oligonucleotide is selected from SEQ ID NO: l-ll. In some embodiments, the antisense oligonucleotide is comprised within a vector system comprising one or more vectors. In some embodiments, the isoform is an Alu element-induced isoform, such as an Alu element-induced TOMM40 isoform. In some embodiments, the TOMM40 isoform comprises the target region set forth in SEQ ID NO: 119. In some embodiments, the subject is a human.
  • the presently disclosed subject matter is directed to a method of measuring the risk of a neurodegenerative disease or a disorder in a subject.
  • the method comprises determining the methylation status of one or more genes of the TOMM complex, TEVIM complex, APOE gene, or associated Alu retrotransposons and comparing the methylation status with a control, thereby identifying the risk of neurodegenerative disease or disorder.
  • determining the methylation status comprises a methylation- specific PCR analysis or methylation- specific digestion analysis.
  • the comparing comprises comparing the methylation status with the status of a confirmed neurodegenerative disease or disorder, or a negative state.
  • the subject is a human.
  • the presently disclosed subject matter is directed to a method of measuring the risk of neurodegenerative disease or disorder in a subject, the method comprising determining the level of A-to-I RNA editing and circular RNA production of one or more genes of the TOMM complex, TEVIM complex, APOE gene, or associated Alu retrotransposons and comparing the A-to-I RNA and circular RNA status with a control, thereby identifying the risk of neurodegenerative disease or disorder. Determining the A-to-I editing and circular RNA status can compress targeted RNA sequencing. The comparing can comprise comparing the A-to-I editing and circular RNA status with the status of a confirmed neurodegenerative disease or disorder, or a negative state. In some embodiments, the subject is a human.
  • the presently disclosed subject matter is directed to a method for treating or inhibiting a neurodegenerative condition caused by one or more TOMM, TIMM, or APOE isoform polynucleotides in the cells of a subject.
  • the method comprises introducing a CRISPR-Cas9 genome editing system into the cells, wherein a Cas9 endonuclease is directed to one or more Alu elements or the regions immediately flanking the one or more Alu elements within theTOMM, TIMM, or APOE isoform polynucleotide in the cells of the subject by a guide RNA to produce an Alu element knockout, an Alu element mutation, or an Alu epigenetic modification.
  • the guide sequence is selected from SEQ ID NOs: 12-118.
  • the subject is a human.
  • Figure 1A is a diagram of human TOMM40 gene (Chromosome 19, GRCh38/h38 assembly; nucleotides 44,891,220-44,903,689) with exons shown as black rectangles.
  • the rs 10524523 genetic variant associated with cognitive impairment, hippocampal thinning, and late-onset Alzheimer's disease is identified. This variant is an Alu element.
  • Figure IB is a diagram of the human TOMM40 gene of Figure la illustrating the Alu elements within introns 6 and 9. A region of enhanced genomic instability arising from Alu element insertion events is identified.
  • Figures 2A and 2B are diagrams representing double stranded RNA structure within TOMM40 intron 6 and intron 9, respectively, resulting from Alu elements arranged in opposite orientation.
  • Figure 3 A is a diagram showing the amino acid sequence of normal functional TOMM40 mRNA. Ribbons indicate the formation of alpha-helices and arrows indicate the formation of beta-strands.
  • Figure 3B is a diagram showing the amino acid sequence of Alu-induced alternative truncated isoform of TOMM40 mRNA resulting in conformational change of the TOMM40 protein. Ribbons indicate the formation of alpha-helices and arrows indicate the formation of beta-strands.
  • Figure 4A is a protein model of normal TOMM40 protein (i.e., Tom40) structure.
  • Figure 4B is a protein model of truncated TOMM40 gene with alternative gene transcripts arising from dysregulation of Alu elements that influence Tom40 protein conformation.
  • FIG. 5 is a model illustrating alternative isoforms of TOMM40 (i.e., Tom40 proteins) cascading into neurodegenerative disease.
  • TOMM40 i.e., Tom40 proteins
  • Figures 6A and 6B are graphs showing elevated Alu insertion events across nuclear- encoded mitochondrial genes within the human genome.
  • Figure 7A is a diagram representing de novo Alu retrotransposition events.
  • Figure 7B is a diagram representing the formation of secondary structures (inverted- repeat Alu duplexes) within pre-mRNA transcripts.
  • Figure 7C is a diagram representing hypomethylation of Alu contributing to exonization. The figure represents both DNA and histone hypomethylation.
  • Figure 8 is a model illustrating how Alu mediated disruption of TOMM and/or TIMM complexes, embedded within the outer and inner mitochondrial membrane, can result in neurodegenerative disease.
  • Figure 8A is a diagram depicting normal function and normal pre -protein transport through the TOMM and TIMM complexes in the outer and inner mitochondrial membrane, respectively.
  • FIG. 8B is a diagram illustrating how Alu mediated disruption of TOMM and/or TIMM complexes can alter pre-protein transport through the outer and/or inner mitochondrial membrane.
  • Alu-mediated disruption of the TOMM and/or TIMM complexes includes deleterious Alu mechanisms that are activated by age or stress-related modification (e.g., traumatic stress) of DNA or histone epigenetic control mechanisms (e.g., altering H3K9 histone methylation). Fluctuating epigenetic landscapes provide tissue-specific and patient- specific deleterious Alu activity.
  • Figure 8C is a diagram of the brain illustrating how deleterious Alu activity cascade into a spectrum of neurodegenerative diseases (e.g., Alzheimer's, ALS, CTE, and Parkinson's disease).
  • neurodegenerative diseases e.g., Alzheimer's, ALS, CTE, and Parkinson's disease.
  • Figure 9A is a diagram representing genes of the TOMM complex that are subject to deleterious Alu activity. Black boxes identify exons within each gene and gray boxes identify Alu elements that introduce genomic instability. Therapeutic target regions are identified by dashed lines. Asterisks identify Alu elements within exons that directly alter protein function. Collectively, the deleterious Alu activity of TOMM genes contributes to mitochondrial dysfunction by destabilizing the translocase of outer mitochondrial membrane (TOMM) complex.
  • TOMM outer mitochondrial membrane
  • Figure 9B is a diagram representing genes of the TIMM complex that are subject to deleterious Alu activity. Black boxes identify exons within each gene and gray boxes identify Alu elements that introduce genomic instability. Therapeutic target regions are identified by dashed lines. Asterisks identify Alu elements within exons that directly alter protein function. Collectively, the deleterious Alu activity of TIMM genes contributes to mitochondrial dysfunction by destabilizing the translocase of inner mitochondrial membrane (TIMM) complex.
  • TIMM inner mitochondrial membrane
  • Figure 9C is a diagram representing the APOE gene that is subject to deleterious Alu activity.
  • Black boxes identify exons within each gene and gray boxes identify Alu elements that introduce genomic instability.
  • Therapeutic target regions are identified by dashed lines.
  • Asterisks identify Alu elements within flanking regions that can influence the epigenetic regulation of APOE.
  • the TOMM40 gene is immediately adjacent to APOE and the Alu epigenetic landscape of TOMM40 can influence the APOE promoter.
  • the term "about”, when referring to a value or to an amount of mass, weight, time, volume, concentration, and/or percentage can encompass variations of, in some embodiments +/-20%, in some embodiments +/- 10%, in some embodiments +1-5%, in some embodiments +/-1%, in some embodiments +1-0.5%, and in some embodiments +/-0.1%, from the specified amount, as such variations are appropriate in the disclosed compositions and methods.
  • the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • mRNA means the presently known mRNA and pre-mRNA transcript(s) of a targeted gene (e.g., TOMM, TIMM, or APOE isoform polynucleotide), and any further transcripts which may be elucidated.
  • a targeted gene e.g., TOMM, TIMM, or APOE isoform polynucleotide
  • antisense oligonucleotide an RNA or DNA molecule that binds to another
  • RNA or DNA e.g., a TOMM, TIMM, or APOE isoform polynucleotide that is mRNA, pre- mRNA, or DNA.
  • RNA or DNA e.g., a TOMM, TIMM, or APOE isoform polynucleotide that is mRNA, pre- mRNA, or DNA.
  • RNA oligonucleotide binds to another RNA target by means of RNA-RNA interactions and alters the activity of the target RNA.
  • An antisense oligonucleotide can upregulate or downregulate expression and/or function of a particular polynucleotide.
  • the definition is meant to include any foreign RNA or DNA molecule which is useful from a therapeutic, diagnostic, or other aspect.
  • Such molecules include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA, and other oligomeric compounds that hybridize to at least a portion of the target isoform polynucleotide.
  • RNAi interference RNA
  • micro RNA micro RNA
  • decoy RNA molecules siRNA
  • enzymatic RNA enzymatic RNA
  • therapeutic editing RNA and other oligomeric compounds that hybridize to at least a portion of the target isoform polynucleotide.
  • these compounds may be introduced in the form of single-stranded, double-stranded, partially single- stranded, or circular oligomeric compounds.
  • the definition is further intended to include linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha- anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • phosphorothioate phosphorothioate
  • methylphosphonate and the like.
  • the antisense oligonucleotides of the present invention can include, but are not limited to, one or a combination of chemical modifications comprising phosphate backbone modifications, phosphorothioate (PS) backbone modification, ribose sugar group modifications, 2'-0-methyl (20Me) modification, 2'-0-methoxy-ethyl (MOE) modification, locked nucleic acid (LNA) modification, tricyclo-DNA (tc-DNA) modification, 2'-fluoro modification, S -constrained-ethyl (cEt) modification, peptide nucleic acid (PNA) modification, or phosphorodiamidate morpholino oligomer (PMO) modification.
  • chemical modifications comprising phosphate backbone modifications, phosphorothioate (PS) backbone modification, ribose sugar group modifications, 2'-0-methyl (20Me) modification, 2'-0-methoxy-ethyl (MOE) modification, locked nucleic acid (LNA) modification, tri
  • the antisense oligonucleotides having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA or pre-mRNA transcript of the targeted gene. Stability of the complexes and duplexes can be determined by theoretical calculations and/or in vitro assays.
  • the term "TOMM, TIMM, or APOE isoform polynucleotide” encompasses DNA, RNA (comprising pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, coding, noncoding sequences, sense or antisense oligonucleotides.
  • RNA comprising pre-mRNA and mRNA
  • cDNA derived from such RNA
  • coding, noncoding sequences sense or antisense oligonucleotides.
  • the specific hybridization of an antisense oligonucleotide with its TOMM, TIMM, or APOE isoform polynucleotide interferes with the normal function of the TOMM, TIMM, or APOE isoform polynucleotide.
  • the functions of DNA to be interfered include, for example, replication and transcription.
  • RNA to be interfered include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.
  • the overall effect of such interference with TOMM, TIMM, or APOE isoform polynucleotide function is modulation of the expression of the encoded product or oligonucleotides.
  • RNA interference "RNAi” is mediated by double stranded RNA (dsRNA) molecules that have sequence-specific homology to their "target" nucleic acid sequences.
  • the mediators are 5-25 nucleotide "small interfering" RNA duplexes (siRNAs).
  • siRNAs small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures that are well known in the art and familiar to the ordinarily skilled artisan.
  • Small interfering RNAs for use in the methods of the present invention suitably comprise between about 1 to about 50 nucleotides (nt).
  • siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.
  • Selection of appropriate antisense oligonucleotides is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products.
  • Translocases are a family of proteins that assist in moving molecules, usually across a membrane.
  • mitochondrial translocases encode channels (e.g., ⁇ -barrel proteins) that are essential for importing proteins into the mitochondria.
  • Mitochondria are present as organelles in eukaryotic cells and produce most of the cell's supply of ATP and are involved in several other processes (i.e., signaling, cellular differentiation, cell death, control of cell division, and cell growth). The stability of mitochondrial translocases is therefore imperative for maintaining functional mitochondria populations within cells.
  • Neurons are especially dependent upon healthy mitochondria populations because they do not undergo active cell division, are highly metabolically active, and require seamless mitochondrial trafficking for survival.
  • Neuronal mitochondrial populations undergo continuous turnover and are renewed though a dynamic process of fusion, fission, and biogenesis. Disruption of the renewal process can result in abnormal function and neuron death.
  • mitochondrial dysfunction is believed to be a major component of multiple neurodegenerative diseases, such as AD, ALS, Parkinson's disease, and Huntington's disease.
  • the molecular mechanisms underlying the origin of mitochondrial dysfunction are believed to include non- mendelian factors, such as tissue-specific epigenetic variation (resulting from stress, traumatic stress, aging, lifestyle factors (e.g., sleep, diet, exercise) or other environmental pressures), retrotransposition of mobile elements resulting in altered gene expression, alternative gene splicing events, and/or the formation of chimeric or fused mRNA products involving genes critical to mitochondrial function.
  • the translocase of the outer mitochondrial membrane is a complex of proteins found on the outer mitochondrial membrane of the mitochondria that allow movement of proteins and pre-proteins into the intermembrane space of the mitochondrion.
  • the TOMM complex includes seven subunits - TOMM5, TOMM6, TOMM7, TOMM20, TOMM22, TOMM40, and TOMM70.
  • TOMM40 forms a ⁇ -barrel protein that is the primary channel through which mitochondrial pre-proteins pass into the mitochondria.
  • the fully assembled TOMM complex comprises three TOMM40 ⁇ -barrel channels arranged in a triangular pattern.
  • the TOMM40 gene is located on human chromosome 19 and encodes 10 exons and nine introns.
  • exon refers to gene regions that are transcribed into RNA and subsequently translated into proteins.
  • intron refers to gene regions that are transcribed into RNA molecules but that are spliced out before the RNA is translated into a protein. Structural variants within TOMM40 introns are statistically associated with cognitive impairment, thinning of the hippocampus, and neurodegenerative disease.
  • the translocase of the inner mitochondrial membrane is a complex of proteins found on the inner membrane of the mitochondria that allow movement of proteins and pre-proteins across the inner mitochondrial membrane and into the mitochondrial matrix.
  • the primary TIMM23 complex forms an import channel that consists of three essential proteins (TEVIM17, TIMM23, and TIMM50) for importing molecules into the inner mitochondrial membrane and, of these, TIMM23 is critical for channel stability.
  • the TIMM23 gene is located on human chromosome 10 (GRCh38/hg38 human genome assembly; ChrlO: nucleotides 45,972,449-46,003,734).
  • the efficient processing of proteins that are central to mitochondrial function depends on stable TOMM and TIMM pore formation and on the seamless trafficking of proteins between the TOMM and TIMM complexes. For this reason, mitochondria are vulnerable to molecular processes that alter TOMM and TIMM stability, especially with respect to conformational changes of the channel forming proteins TOMM40 and TIMM23.
  • the presently disclosed subject matter focuses on disrupting those processes involving retrotransposons and/or mRNA fusion events that serve to degrade mitochondrial function in neurons and contribute to the development of sporadic neurological and neurodegenerative disorders.
  • Retrotransposons are mobile elements that can replicate by reverse transcription of an RNA intermediate and insert themselves into new locations across the genome.
  • retrotransposons There are three classes of retrotransposons: long terminal repeats (LTRs), long interspersed elements (LINEs), and short interspersed elements (SINEs).
  • LTRs long terminal repeats
  • LINEs long interspersed elements
  • SINEs short interspersed elements
  • Alu elements are a highly successful primate- specific SINE and are the most abundant mobile element in the human genome, having over a million copies that comprise approximately 11% of genomic DNA. Initially considered "junk DNA", Alu elements are known to have profound impacts on gene splicing, gene expression, and gene stability. In addition, Alu elements have been implicated in a growing number of human diseases, including neurological disorders.
  • Alu elements include a high percentage of CpG islands (i.e., short stretches of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases in a 5' to 3' direction) and are typically heavily methylated (both DNA and histone H3K9 methylation) to prevent their expression.
  • Alu elements are approximately 300 nucleotides in length and are characterized by left and right monomers with a poly- A tail at their 3' end. Multiple subfamilies of Alu elements have been identified within humans (i.e., AluJ, AluS, AluY) and variable rates of retrotransposition have been observed across the subfamilies.
  • Alu elements residing within genes are transcribed by RNA polymerase II and are a part of pre- mRNA transcripts.
  • Alu elements within pre-mRNA molecules are subject to enhanced epigenetic RNA editing (e.g., adenosine-to-inosine (A-to-I) editing) by adenosine deaminase acting on RNA (ADAR) proteins.
  • RNA editing e.g., adenosine-to-inosine (A-to-I) editing
  • ADAR adenosine deaminase acting on RNA
  • Alu element-rich regions are susceptible to Alu element-mediated genome instability, alternative splicing events that alter protein structure/function, inhibitory pre-mRNA structures, retention and degradation of heavily A-to-I edited pre-mRNAs, and production of chimeric protein products that interfere with key cellular processes.
  • Figure 1A is a diagram of the human TOMM40 gene (GRCh38/hg38 assembly; Chrl9 nucleotides 44,891,220 - 44,903,689) with exons shown as grey rectangles. Primate- specific Alu retrotransposons have repeatedly inserted into TOMM40 introns.
  • Figure 1A also shows a variable deoxythymidine homopolymer repeat (poly-T; variant rs 10524523, statistically associated with cognitive impairment, hippocampal thinning, and late-onset Alzheimer's disease risk) that is part of an Alu element and originated from an Alu insertion event.
  • poly-T variable deoxythymidine homopolymer repeat
  • variant rs 10524523 variant rs 10524523
  • the dashed line below the gene diagram identifies the primary region of enhanced genomic instability of the TOMM40 gene associated with Alu elements.
  • Age, stress, lifestyle factors (e.g, sleep, diet, exercise), and other environment related epigenetic modification (e.g., both hyper- and hypomethylation), de novo Alu element insertion events, excessive Alu A-to-I RNA editing, introduction of premature stop codons, and/or exonization of Alu elements within the region is believed to contribute to either alternative mRNA splicing events of TOMM40 or increased degradation of TOMM40 mRNAs.
  • the arrows in Figure IB show Alu element orientation of insertion events across TOMM40. Alu elements having opposite orientation contribute to double stranded RNA structures impacting TOMM40 gene expression.
  • Figures 2A and 2B illustrate one embodiment of the double stranded RNA structure within TOMM40 intron 6 and TOMM40 intron 9, respectively, resulting from Alu elements arranged in opposite orientations.
  • TOMM40 introns 6 and 9 contribute to transcriptional noise through elevated non-sense mediated decay (e.g., due to enhanced A-to-I editing) and/or the production of alternative TOMM40 isoforms.
  • Genetic variants of TOMM40 have been associated with dementia and neurodegenerative disease. Specifically, both single nucleotide polymorphisms (SNPs) and short structural variants (SSV) within TOMM40 have been implicated in a number of neurological disorders, ranging from mild cognitive impairment to major neurodegenerative diseases (such as LOAD and PD). Disrupting the TOMM complex effectively triggers a mitochondrial stress response and can ultimately lead to inflammation and mitophagy, a process that exhibits devastating consequences across neurological networks.
  • SNPs single nucleotide polymorphisms
  • SSV short structural variants
  • tissue-specific aging, stress, lifestyle factors (e.g., sleep, diet, exercise) and/or environmentally-associated epigenetic modification of Alu elements (i.e., both DNA and histone H3K9 methylation or acetylation) within TOMM40 introns 6 and/or 9 is believed to contribute to an increase of alternative splicing events of TOMM40 and/or to increased production of modified TOMM40 proteins thereby contributing to the establishment of dysfunctional TOMM pores (resulting in protein aggregate accumulation) and/or decreased mitochondrial populations in neurons.
  • the Alu element-rich 3' region of TOMM40 is unstable due to multiple Alu element insertion events that can contribute to the formation of double- stranded pre-mRNA structures (including circRNAs) and enhanced A-to-I editing of pre-mRNA molecules that effectively alters TOMM40 gene expression leading to mitochondrial stress and dysfunction.
  • Inverted-repeat Alu elements such as those distributed across TOMM40 introns 6 and 9, can disrupt mRNA stability by facilitating premature transcription termination and altering A-to-I RNA editing.
  • the Alu elements enriched across the 3' end of TOMM40 are believed to contribute to the production of modified yet functional transcripts that escape nonsense-mediated mRNA decay and directly alter the TOMM40 protein conformation.
  • three Alu elements within TOMM40 intron 9 are associated with an alternative splicing event that results in a mRNA product of 335 amino acids in length, 26 amino acids shorter than the normal TOMM40 mRNA product.
  • Figure 3 A is a diagram showing the amino acid sequence of normal functional TOMM40 mRNA (NCBI GenBank accession number CP_006105; 361 amino acids in length; protein strands and helices are shown in arrows and spirals).
  • Figure 3B is a diagram showing the amino acid sequence of Alu-induced alternative truncated isoform of TOMM40 mRNA (NCBI GenBank accession XM_005258468; 335 amino acids in length).
  • FIG. 4A is a 3-dimensional model of the normal TOMM40 protein structure.
  • Figure 4B is a predictive 3-dimensional model showing the potential influence of premature termination of the TOMM40 gene transcript on the TOMM40 protein structure.
  • the 3' end of the truncated 335 amino acid transcript coincides with the AluY retrotransposon within intron 9 of TOMM40 (as shown in Figure 1).
  • the blue shaded regions identify major conformational changes to the ⁇ -barrel protein. Without being restricted to any particular mechanism of action, it is proposed that conformational changes of TOMM40 may contribute to the initial formation of inflammatory protein bodies observed in neurodegenerative disease.
  • Alu element-associated disruption of normal mRNA processing of TOMM40 may result in increased localization of the paralog TOMM40L to the mitochondrial outer membrane.
  • the efficient processing of mitochondrial pre-proteins can be modified and/or the TOMM complex can be otherwise destabilized.
  • Such a mechanism could result in the propagation of inefficient TOMM channels through mitochondrial biogenesis, fusion, and fission within individual neurons over variable time-scales, as shown in Figure 5. This mechanism can account for the initial establishment and accumulation of intra-cellular protein bodies that are the hallmark of multiple neurodegenerative disorders.
  • TOMM5 Alu element exonization and somatic retrotransposition events of both LI and Alu elements have been identified in multiple TOMM genes (i.e., TOMM5, TOMM7, TOMM22, TOMM40, TOMM40L), indicating that TOMM genes are actively influenced by and are vulnerable to retrotransposons, likely owing to their high transcription rates and open chromatin status.
  • TOMM genes are actively influenced by and are vulnerable to retrotransposons, likely owing to their high transcription rates and open chromatin status.
  • the transcriptionally active -2,000 nuclear-encoded genes that are essential for mitochondrial function are especially vulnerable to retrotransposon disruption, particularly within energetically demanding neurons. Given this hypothesis, it would be expected that nuclear-encoded mitochondrial genes would display an enrichment of mobile elements with respect to other genes.
  • the human genome has evolved a number of mechanisms to defend against deleterious retrotransposon activity, including DNA methylation, histone methylation, and RNA degradation using miRNA-processing enzymes.
  • epigenetic silencing is predominantly mediated by histone (H3K9) methylation to suppress transcription and retrotransposition.
  • Hypomethylation (loss of the methyl group in the 5-methylcytosine nucleotide) contributes to enhanced retrotransposon activity, which in turn can increase transcriptional noise by disrupting gene expression pathways, inducing alternative splicing events and reducing mRNA stability.
  • Genome-wide Alu element hypomethylation is part of the aging process, and global hypomethylation of Alu elements has been statistically associated with Alzheimer's disease, multiple sclerosis, osteoporosis, and many forms of cancer.
  • both DNA and histone methylation landscapes can be altered by both lifestyle factors (e.g., sleep, diet, exercise) and traumatic stress events (e.g., brain injury arising from concussion).
  • lifestyle factors e.g., sleep, diet, exercise
  • traumatic stress events e.g., brain injury arising from concussion.
  • Epigenetic modification including both hyper- and hypomethylation
  • Alu elements and/or de novo Alu element insertions within (or in close proximity to) genes that are essential to mitochondrial function are believed to contribute to mRNA instability, ultimately leading to mitochondrial dysfunction.
  • retrotransposons can influence gene splicing by de novo Alu element retrotransposon events (as illustrated in Figure 7A), through the formation of inverted repeat Alu element duplexes within pre-mRNA transcripts (as illustrated in Figure 7B), and through hypomethylation of Alu elements that contribute to exonization (as illustrated in Figure 7C).
  • histone H3K9 also regulates APOE transcription.
  • APOE apolipoprotein E
  • APOE apolipoprotein E
  • Variants of the APOE gene are statistically associated with Alzheimer's disease onset.
  • the Alu element-rich regions of TOMM40 are immediately upstream of APOE on human chromosome 19.
  • the epigenetic interaction between Alu elements within TOMM40 and the APOE promoter is believed to influence TOMM40 gene expression and/or APOE gene expression.
  • TOMM40 gene expression and/or APOE gene expression is believed to be an epigenetic link between time-dependent mitochondrial dysfunction (both slowly accumulating or accelerated) and tissue- specific idiopathic neurodegenerative disease.
  • Recent data from single-cell genome sequencing provides evidence of unique or mosaic genomes of individual neurons arising from enhanced retrotransposon activity both during neurogenesis and throughout life.
  • individual neurons can have differing intracellular conditions that influence gene transcription, translation, and protein formation.
  • Mitochondrial genes encoded within the nuclear genome are particularly vulnerable to enhanced retrotransposon activity (including elevated Alu-based epigenetic RNA editing and production of Alu-based circular RNAs) given their high expression and open chromatin status within neurons. This vulnerability increases with age, stress, lifestyle factors (e.g., sleep, diet, exercise), and/or environmental conditions that alter the epigenetic landscape of neurological networks (as described above).
  • retrotransposons operating though human-specific neurological pathways, are believed to contribute to environment, stress, lifestyle, and/or age-related neurodegeneration by disrupting functional mitochondrial populations within neurons (Figure 8).
  • the mitochondrial disruption can occur through a number of retrotransposon-induced mechanisms that can influence the efficient and accurate transcription and/or translation of the -2,000 mitochondrial genes encoded in the nuclear genome ( Figures 6 A and 6B).
  • TOMM40 it is believed that Alu element-related conformational changes (both subtle and major) of the outer and inner mitochondrial membrane pores restrict or prevent the normal translocation of proteins (i.e., TOMM and TIMM complexes), ultimately contributing to mitochondrial stress and mitophagy.
  • the vulnerability can be amplified through mitochondrial biogenesis and downstream mitochondrial fission and fusion events, thus contributing to the initial establishment of inefficient mitochondria that increase mitochondrial stress over time, contribute to the formation of protein aggregates, and limit neuron functionality, ultimately cascading into a diseased state (Figure 8).
  • tissue-specific cascade events can manifest in different neurological tissues.
  • tissue-specific cascade events operating through variable inter-cellular and intra-cellular conditions and occurring at different life stages, can ultimately result in diseased states that share similar underlying pathologies with patients displaying a spectrum of neurological impairments.
  • tissue-specific deleterious cascade events can originate from traumatic stress events known to disrupt retrotransposon epigenetic control mechanisms (including physical and emotional trauma).
  • Alu-induced protein isoforms can be reduced using antisense therapy (i.e., mRNA antisense oligonucleotides).
  • An "antisense" oligonucleotide comprises a nucleotide sequence that is complementary to a "sense" nucleic acid, e.g., complementary to the mRNA sequence of a target. Once the antisense oligonucleotide binds to the target mRNA, the associated gene is inactivated (i.e., turned off) because mRNA must be single stranded for translation to occur.
  • the target mRNA can be can be an Alu element- induced isoform of TOMM and TIMM gene products, or the APOE gene.
  • the isoform can be an Alu element-induced TOMM40 isoform.
  • the term "isoform" as used herein refers to an analog or mutant having one or more amino acid modifications at one or more amino acid positions of a natural fully-functional human protein.
  • the antisense oligonucleotides can span both normal sequences and Alu element sequences within TOMM, TIMM, or APOE isoform mRNA.
  • the disclosed antisense oligonucleotides can be administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA encoding the isoform to thereby inhibit translation.
  • the hybridization can be through conventional nucleotide complementarity to form a stable duplex.
  • the antisense nucleic acid molecule can be administered systemically and modified to target selected cells.
  • an antisense molecule can be modified such that it specifically binds to a TOMM, TIMM, or APOE isoform on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody that binds to a cell surface receptor or antigen.
  • the antisense nucleic acid molecule can also be delivered to cells using a vector system, as would be known to those of ordinary skill in the art.
  • the presently disclosed subject matter is directed to a composition comprising one or more antisense oligonucleotides specific for an isoform of TOMM, TIMM, or APOE polynucleotide.
  • a pharmaceutically acceptable amount of the disclosed antisense oligonucleotides is administered.
  • pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable (i.e., the material can be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition).
  • the presently disclosed subject matter can comprise methods of modulating the function and/or expression of a TOMM, TIMM, or APOE isoform in a subject's cells or tissues.
  • the modulation can be in vivo or in vitro.
  • the method comprises contacting the cells or tissues of a subject (i.e., a human) with at least one antisense oligonucleotide.
  • the presently disclosed subject matter is directed to a method of preventing or treating a disease or disorder associated with at least one TOMM, TIMM, or APOE isoform.
  • the method comprises administering to a subject a therapeutically effective dose of a composition comprising at least one antisense oligonucleotide that binds to a TOMM, TIMM, or APOE isoform polynucleotide, thereby preventing or treating the disease or disorder.
  • the disease or disorder is a neurological disorder, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or Parkinson's disease.
  • the antisense oligonucleotides of the present disclosure have at least about 50% sequence identity (i.e., at least about 50, 55, 60, 65, 70, 75, 80 ,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9%) to a reverse complement of an isoform of TOMM, TEVIM, or APOE, such that function and/or expression of the isoform polynucleotide is modulated in the cells/tissue of the subject.
  • sequence identity i.e., at least about 50, 55, 60, 65, 70, 75, 80 ,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9%
  • Table 1 illustrates nucleotide sequences of human TOMM40 antisense oligonucleotides targeting TOMM40 intron 9.
  • Therapeutic targets include Alu-associated TOMM40 messenger RNA products.
  • Application of antisense oligonucleotides can mediate alternative splicing events of TOMM40 by binding to and eliminating mRNA products that include retained intron 9 or exons extending into Alu retrotransposon sequences within intron 9.
  • Table 2 illustrates antisense oligonucleotides targeting TOMM40L (the functional paralog of TOMM40; also identified as TOMM40B) to prevent enrichment of TOMM40L proteins in neuron mitochondrial populations.
  • Therapeutic targets consist of nine TOMM40L exons and application of antisense oligonucleotides can reduce mRNA expression of TOMM40L, thereby stabilizing the central beta-barrel channels of the translocase of outer mitochondrial membrane pore.
  • Table 3 illustrates nucleotide coordinates for germline insertions of Alu retrotransposons in TOMM40 and APOE on human chromosome 19 (build GRCh38 / hg38).
  • Age-related or environmentally induced demethlylation of Alu retrotransposons contributes to alternative splicing events, transcriptional noise, and genomic instability of TOMM40 and/or APOE.
  • Targeted epigenetic modification (including DNA and/or histone modification) of Alu retrotransposons within TOMM40 and/or APOE, and associated intergenic regions, using the CRISPR-Cas9 can result in the stabilization of TOMM40 and/or APOE gene transcription.
  • the antisense oligonucleotides described herein can comprise any pharmaceutically acceptable salts, esters, or any other functional chemical equivalent that, upon administration to subject (e.g., a human) is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • subject e.g., a human
  • the presently disclosed subject matter includes prodrugs and pharmaceutically acceptable salts of the antisense oligonucleotides disclosed herein, pharmaceutically acceptable salts of such prodrugs, and other bio equivalents.
  • prodrug refers a therapeutic agent that is prepared in an inactive or less active form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes, chemicals, and/or conditions.
  • the term "pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the disclosed compounds (i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto).
  • sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans.
  • the disclosed antisense oliogonucleotides can be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures, or mixtures of compounds.
  • antisense oligonucleotides described herein can comprise one or a combination of chemical modifications to enhance stability against degradation by endo- and exonucleases and/or to improve targeting to the cells of interest. Methods for such improvement of the pharmacological profile of antisense oligonucleotides are known to those of skill in the art (see, e.g., Evers et al. (2015) Advanced Drug Delivery Reviews 87:90-103). For example, antisense oligonucleotides can be prepared having a range of different modifications on the phosphate backbone and ribose sugar group in the case of RNA.
  • the antisense oligonucleotides of the present invention can include, but are not limited to, one or a combination of chemical modifications comprising phosphate backbone modifications, phosphorothioate (PS) backbone modification, ribose sugar group modifications, 2'-0-methyl (20Me) modification, 2'-0-methoxy-ethyl (MOE) modification, locked nucleic acid (LNA) modification, tricyclo-DNA (tc-DNA) modification, 2'-fluoro modification, S -constrained-ethyl (cEt) modification, peptide nucleic acid (PNA) modification, or phosphorodiamidate morpholino oligomer (PMO) modification.
  • chemical modifications comprising phosphate backbone modifications, phosphorothioate (PS) backbone modification, ribose sugar group modifications, 2'-0-methyl (20Me) modification, 2'-0-methoxy-ethyl (MOE) modification, locked nucleic acid (LNA) modification, tri
  • the antisense oligonucleotides of the present invention can be administered intraventricularly, intranasally, intrathecally, or systemically to the subject.
  • the antisense oligonucleotides are tagged with a cell-penetrating peptide (CPP) for systemic administration, such as intravenous administration, in order to allow for passing of the blood brain barrier.
  • CPP cell-penetrating peptide
  • the antisense oligonucleotides are encapsulated in exosomes for systemic administration, such as intravenous administration, in order to allow for passing of the blood brain barrier.
  • the presently disclosed subject matter is directed to a method of measuring, reducing, and/or eliminating the risk of mitochondrial dysfunction and/or neurodegenerative disease by modifying the Alu elements and/or regions immediately flanking Alu elements within one or more genes of the TOMM complex (including the paralog of TOMM40, TOMM40L), one or more genes of the TIMM protein complex, and/or Apolipoprotein E (APOE).
  • the Alu elements and flanking regions can be modified by epigenetic regulators (e.g. DNA and/or histone epigenetic activators and/or repressors), targeted DNA mutation, and targeted excision, or combinations thereof.
  • targeted bioassays of the methylation status of one or more genes of the TOMM complex, one or more genes of the TIMM complex, the APOE gene, and/or associated Alu retrotransposons can be performed to determine neurodegenerative disease risk or progression status.
  • Human genome coordinates (build GRch38/hg38) for Alu biomarker/epimarker analyses to determine neurodegenerative disease risk are provided in Table 7.
  • the Alu elements within or adjacent to genes encoding proteins of the TOMM and/or TIMM complexes and/or the APOE gene can be epigenetically modified or disrupted using a therapy comprising of clustered regularly interspaced short palindromic repeats (CRISPPv)-Cas genome editing technology that can target specific DNA sequences within a given genome.
  • CRISPR-Cas9 includes the use of a Cas9 protein and one or more guide RNAs targeting the desired sequence.
  • the target sequence is contacted with a CRISPR-associated Cas protein and 1-2 ribonucleic acids, where the ribonucleic acids direct the Cas protein to and hybridize to the target.
  • CRISPR-Cas9 mediated epigenetic repression of Alu elements is achieved by utilizing short (-14 base pairs) single-guide (or small- guide) RNA (sgRNA) sequences that bind to DNA regions immediately flanking Alus. When combined with CRISPR-Cas9 these short sgRNA allow for Cas9 binding without cleaving the target locus. Binding of the Cas9 complex serves to repress Alu activity.
  • sgRNA single-guide (or small- guide) RNA
  • epigenetic repression of Alu elements is achieved using CRISPR/dCas9-KRAB and other chromatin-modifying enzymes with guide RNA sequences targeting Alu elements within or neighboring TOMM, TIMM, and/or the APOE gene.
  • CRISPR-Cas9 DNA mutation or excision of Alu elements within or neighboring TOMM, TIMM, and/or the APOE gene is achieved by inducing double-strand breaks that are then repaired by native non-homologous end joining or homology-directed repair pathways, to those regions immediately flanking Alu elements.
  • CRISPR-Cas9 guide RNAs targeting those flanking regions are provided below.
  • Table 1 illustrates nucleotide sequences of human TOMM40 antisense oligonucleotides targeting TOMM40 intron 9.
  • Therapeutic targets include Alu-associated TOMM40 messenger RNA products.
  • Application of antisense oligonucleotides can mediate alternative splicing events of TOMM40 by binding to and eliminating mRNA products that include retained intron 9 or exons extending into Alu retrotransposon sequences within intron 9.
  • Table 2 illustrates antisense oligonucleotides targeting TOMM40L (the functional paralog of TOMM40; also identified as TOMM40B) to prevent enrichment of TOMM40L proteins in neuron mitochondrial populations.
  • Therapeutic targets consist of nine TOMM40L exons and application of antisense oligonucleotides can reduce mRNA expression of TOMM40L, thereby stabilizing the central beta-barrel channels of the translocase of outer mitochondrial membrane pore.
  • Table 3 illustrates nucleotide coordinates for germline insertions of Alu retrotransposons in TOMM40 and APOE on human chromosome 19 (build GRCh38 / hg38).
  • Age-related or environmentally induced demethlylation of Alu retrotransposons contributes to alternative splicing events, transcriptional noise, and genomic instability of TOMM40 and/or APOE.
  • Targeted epigenetic modification (including DNA and/or histone modification) of Alu retrotransposons within TOMM40 and/or APOE, and associated intergenic regions, using the CRISPR-Cas9 can result in the stabilization of TOMM40 and/or APOE gene transcription.
  • CRIS PR-Guide sequences for the 5' and 3' modification of the Alu complex flanking the rs 10524523 polymorphism within TOMM40 intron 6 are provided in Table 4.
  • CRISPR-Guide sequences for modification of Alu elements within TOMM40 intron 9 are provided in Table 5.
  • the TIMM23/TOMM40 fusion mRNA antisense oligonucleotide therapy target region is set forth in Table 6.
  • the transcriptome, genome, and epigenome of an organism can be manipulated using targeted antisense oligonucleotide and/or CRISPR/Cas9 or technologies or another technology similar to CRISPR/Cas9.
  • the presently disclosed subject matter includes composition that can be administered to a subject.
  • Therapeutic delivery of antisense oligonucleotides and/or CRISPR- components that target Alu related isoforms of TOMM, TIMM, or APOE genes and/or regions flanking Alu elements can include, but are not limited to, viral vectors including adeno-associated virus (AAV), lipid-mediated delivery including liposomes and/or related lipid nanoparticles, nanogels, electroporation, and polyethylenimine mediated transfection.
  • AAV adeno-associated virus
  • lipid-mediated delivery including liposomes and/or related lipid nanoparticles, nanogels, electroporation, and polyethylenimine mediated transfection.
  • the presently disclosed subject matter is directed to a therapeutic genome editing method for treating or inhibiting a condition (such as a neurological condition) caused by Alu retrotransposons related to TOMM, TIMM, or APOE genes and proteins.
  • a method is provided of modulating the function, expression, or both of a TOMM, TIMM, or APOE isoform polynucleotide in the cells of a subject, the method comprising: contacting said cells with an antisense oligonucleotide, wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of a TOMM, TIMM, or APOE isoform polynucleotide, thereby modulating function, expression, or both of the isoform.
  • the modulating can occur in vivo or in vitro.
  • the antisense oligonucleotide can be selected from SEQ ID NOs: l-ll.
  • the antisense oligonucleotide can be comprised within a vector system comprising one or more vectors.
  • the antisense oligonucleotide can have at least 75% sequence identity to a reverse complement of a TOMM, TIMM, or APOE isoform polynucleotide.
  • the antisense oligonucleotide can have at least 95% sequence identity to a reverse complement of a TOMM, TIMM, or APOE isoform polynucleotide.
  • the TOMM, TIMM, or APOE isoform polynucleotide can be an Alu element-induced isoform.
  • the TOMM, TIMM, or APOE isoform polynucleotide can be an Alu element-induced TOMM40 isoform.
  • the TOMM40 isoform can comprise the target region set forth in SEQ ID NO: 119.
  • the subject can be a human.
  • TOMM and TIMM genes, and/or the APOE gene can be stabilized or restored using histone deacetylases inhibitors (HDACis) and/or thiazolidinedione compounds (TZDs) comprised of short chain fatty acids, hydroxamic acids and benzamides including but not limited to nicotinamide, VPA, sodium butyrate, TSA, SAHA, phenylbutyrate, Ex527, and benzamide ms- 275.
  • HDACis histone deacetylases inhibitors
  • ZTDs thiazolidinedione compounds
  • Therapeutic delivery mechanisms of Alu stabilizing HDACis and TZDs can include, but are not limited to, oral delivery, cerebrospinal delivery via lumbar puncture, lipid-mediated delivery including liposomes and/or related lipid nanoparticles, nanogels, electroporation, polymer-based particles, and polyethylenimine mediated transfection.
  • epigenetic processes centered on Alu elements within TOMM, TIMM, and APOE pre-mRNAs and mRNAs can be measured for biomarker/epimarker analyses using targeted high-throughput sequencing technologies (e.g., ILLUMINA TECHNOLOGIES), targeted Sanger sequencing, and/or targeted single-molecule sequencing (e.g., PACIFIC BIOSCIENCES and/or OXFORD NANOPORE TECHNOLOGIES). These processes include, but are not limited to, A-to-I editing levels and/or presence or absence of circular RNAs associated with TOMM, TIMM, and/or APOE genes. Results from pre-mRNA biomarker analyses can be used to determine or quantify neurodegenerative disease risk with respect to epigenetic Alu-related pathways discussed herein.
  • DNA and/or histone methylation status of Alu elements within or neighboring TOMM and TIMM genes, and/or the APOE gene can be measured for biomarker/epimarker analyses using targeted bisulfite-sequencing, chromatin immunoprecipitation high-throughput sequencing (ChlP-seq), single-molecule real-time sequencing (e.g. Pacific Biosciences Technologies), and/or single-molecule nanopore sequencing (e.g. Oxford Nanopore Technologies).
  • Targeted epimarker analyses can be performed using baits or probes designed to bind to conserved regions within TOMM and TIMM genes and/or the APOE gene (coordinates provided in Table 3).
  • the biological samples required for biomarker/epimarker analyses would include peripheral blood and/or cerebrospinal fluids.
  • the DNA and/or histone methylation status of Alu elements within and/or neighboring TOMM, TIMM, and/or the APOE gene can be used to measure and/or predict Alu-related destabilization of genes essential to mitochondrial function.
  • biomarker/epimarker analyses of Alu elements within TOMM, TIMM, and/or APOE gene focus on individual or patient- specific Alu variation arising from maternal, paternal, and/or de novo insertion events and include phased analyses. Results from the Alu biomarker/epimarker assay are used to determine or quantify neurodegenerative disease risk with respect to the Alu-related molecular pathways discussed herein.
  • the method can be carried out in vivo or ex vivo.
  • the presently disclosed subject matter is not limited to neuronal mitochondrial dysfunction and neurodegenerative disease.
  • mitochondrial dysfunction can underlie the origin of a wide variety of human diseases including (but not limited to) cancer (e.g., prostate, liver, brain, colon, lung, gastric, breast, leukemia, ovary, thyroid, salivary, goiter, kidney), cardiovascular disease (e.g., atherosclerosis, ischemic heart disease, heart failure, hypertension), metabolic disease (e.g., insulin resistance, diabetes, obesity-associated metabolic syndrome, dysglycemia, dyslipidemia), inflammatory disease, and osteoporosis. Therefore, the compositions and methods provided herein to interfere with the retrotransposon or Alu-mediated molecular mechanisms applicable to nuclear-encoded mitochondrial genes associated with a broad range of human diseases.
  • Alu-related molecular mechanisms that can disrupt the efficient transcription and/or translation of nuclear-encoded mitochondrial genes can include (but are not limited to) de novo Alu insertion, Alu exonization, altered RNA editing, altered adenosine-to-inosine (A-to-I) editing, premature gene translation termination, alternative gene splicing, nonhomologous recombination events, altered micro-RNA regulation, altered binding of long non-coding RNAs, hypomethylation, and hypermethylation.
  • the disruptive Alu mechanisms are a plausible source for human disease wherein incipient or early mitochondrial dysfunction is hypothesized.
  • epigenetic control mechanisms of Alu elements dissipates over time and hypomethylation of Alu elements is associated with aging and senescence.
  • Alu-mediated disruption of nuclear-encoded mitochondrial genes potentially correlates with the aging process and is directly linked with age-related human disease.
  • Therapeutic intervention of Alu-associated mechanisms operating on nuclear-encoded mitochondrial genes can be achieved by the delivery of targeted antisense oligonucleotides, modification of Alu epigenetic profiles (including flanking nucleotides; e.g., using CRISPR/Cas technology and/or pharmaceutical compounds that alter DNA and/or histone methylation), targeted excision or removal of Alu elements (e.g., using CRISPR/Cas technology), as well as the delivery of agents that alter mitochondrial biogenesis, fusion, or fission.
  • the cited therapeutic approaches are for stabilization of gene transcription and translation (including stabilization of pre-mRNA molecules and mature RNA transcripts) of nuclear-encoded mitochondrial genes that are influenced by the presence of Alu mobile elements.
  • Therapeutic modification of Alu elements within TOMM40 can be achieved using mRNA antisense oligonucleotides, targeted CRISPR-Cas9 methylation, and/or CRISPR-Cas9 removal of Alu elements in intron 6 and/or intron 9.
  • CRISPR-Cas9 targeting for targeted methylation and/or excision of Alu elements can be performed between bases 44,894,113 and 44,900,752 on human chromosome 19 (UCSC Genome Browser; assembly GRCh38/hg38).
  • CRISPR-Cas9 targeting for targeted methylation and/or excision of Alu elements can be performed between bases 44,901,330 and 44,903,034 on human chromosome 19 (UCSC Genome Browser; assembly GRCh38/hg38).
  • Antisense oligonucleotides to reduce alternative Alu-induced TOMM40 isoforms can be designed to dually span both normal exons and Alu sequences within TOMM40 mRNA.
  • Table 1 illustrates nucleotide sequences of human TOMM40 antisense oligonucleotides targeting TOMM40 intron 9.
  • Therapeutic targets include Alu-associated TOMM40 messenger RNA products.
  • Application of antisense oligonucleotides can mediate alternative splicing events of TOMM40 by binding to and eliminating mRNA products that include retained intron 9 or exons extending into Alu retrotransposon sequences within intron 9.
  • Table 2 illustrates antisense oligonucleotides targeting TOMM40L (the functional paralog of TOMM40; also identified as TOMM40B) to prevent enrichment of TOMM40L proteins in neuron mitochondrial populations.
  • Therapeutic targets consist of nine TOMM40L exons and application of antisense oligonucleotides can reduce mRNA expression of TOMM40L, thereby stabilizing the central beta-barrel channels of the translocase of outer mitochondrial membrane pore.
  • Table 3 illustrates nucleotide coordinates for germline insertions of Alu retrotransposons in TOMM40 and APOE on human chromosome 19 (build GRCh38 / hg38).
  • Age-related or environmentally induced demethlylation of Alu retrotransposons contributes to alternative splicing events, transcriptional noise, and genomic instability of TOMM40 and/or APOE.
  • Targeted epigenetic modification (including DNA and/or histone modification) of Alu retrotransposons within TOMM40 and/or APOE, and associated intergenic regions, using the CRISPR-Cas9 can result in the stabilization of TOMM40 and/or APOE gene transcription.
  • CRIS PR-Guide sequences for the 5' and 3' modification of the Alu complex flanking the rs 10524523 polymorphism within TOMM40 intron 6 are provided in Table 4.
  • CRISPR-Guide sequences for modification of Alu elements within TOMM40 intron 9 are provided in Table 5.
  • the TIMM23/TOMM40 fusion mRNA antisense oligonucleotide therapy target region is set forth in Table 6.
  • DNA and or histone methylation status of Alu elements within or neighboring nuclear- encoded mitochondrial genes including but not limited to genes of the TOMM and TIMM complexes, and neighboring genes implicated in neurodegenerative disease manifestation (e.g. APOE) are measured using both system-wide whole genome sequencing approaches (e.g., bisulfate-sequencing and/or chromatin immunoprecipitation high-throughput sequencing) and targeted approaches with probes or baits designed to bind to and select regions or genes of interest (relevant coordinates provided in Table 7).
  • Detection of elevated DNA and/or histone demethylation (i.e., enhanced or progressive hypomethylation) of Alu mobile elements within biological samples are used to measure neurodegenerative disease risk and inform downstream therapeutic approaches.
  • Personalized baseline biomarker/epimarker data generated from biological samples e.g., peripheral blood, cerebrospinal fluids
  • biological samples e.g., peripheral blood, cerebrospinal fluids
  • the resulting biomarker/epimarker data are used to measure neurodegenerative disease risk within patients across time.
  • AluSx chr3 100380064-10038036
  • AluSx 1 chr3 100395404-100395572 AluSg chr3: 100397609-100397899
  • TIMM17 1 201,955,491 - 201,970,661
  • TIMM50 19 39,480,412 - 39,490,888

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne les rétrotransposons, fonctionnant via des voies neurologiques spécifiques à l'homme, qui peuvent contribuer à l'environnement, au style de vie et/ou à la neurodégénérescence liée à l'âge par la perturbation de populations mitochondriales fonctionnelles dans les neurones. Le dysfonctionnement mitochondrial peut se produire via un certain nombre de mécanismes induits par des rétrotransposons qui peuvent influencer la transcription et/ou la traduction efficace(s) et précise(s) de gènes mitochondriaux encodés dans le génome nucléaire, fonctionnant principalement par des processus épigénétiques. Des changements conformationnels liés à l'élément alu (à la fois subtils et majeurs) des pores de la membrane mitochondriale externe et interne peuvent restreindre ou empêcher la translocation normale de protéines (c'est-à-dire des complexes TOMM et TIMM), ce qui contribue finalement au stress mitochondrial, à la mitophagie, à l'inflammation et à la mort des neurones et des cellules gliales. L'invention concerne des compositions et des procédés pour atténuer et/ou prévenir des changements conformationnels induits par un élément Alu afin de prévenir et/ou de traiter une maladie neurodégénérative et d'autres maladies et troubles associés à au moins une isoforme TOMM, TIMM ou APOE comprenant le cancer et d'autres maladies inflammatoires.
PCT/US2018/013645 2017-01-12 2018-01-12 Compositions et procédés pour perturber les mécanismes moléculaires associés à un dysfonctionnement mitochondrial et à une maladie neurodégénérative WO2018132755A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/477,645 US11352625B2 (en) 2017-01-12 2018-01-12 Compositions and methods for disrupting the molecular mechanisms associated with mitochondrial dysfunction and neurodegenerative disease
US17/723,555 US11932855B2 (en) 2017-01-12 2022-04-19 Compositions and methods for disrupting the molecular mechanisms associated with mitochondrial dysfunction and neurodegenerative disease

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201762445279P 2017-01-12 2017-01-12
US62/445,279 2017-01-12
US201762468146P 2017-03-07 2017-03-07
US62/468,146 2017-03-07

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US16/477,645 A-371-Of-International US11352625B2 (en) 2017-01-12 2018-01-12 Compositions and methods for disrupting the molecular mechanisms associated with mitochondrial dysfunction and neurodegenerative disease
US17/723,555 Division US11932855B2 (en) 2017-01-12 2022-04-19 Compositions and methods for disrupting the molecular mechanisms associated with mitochondrial dysfunction and neurodegenerative disease

Publications (1)

Publication Number Publication Date
WO2018132755A1 true WO2018132755A1 (fr) 2018-07-19

Family

ID=62840188

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/013645 WO2018132755A1 (fr) 2017-01-12 2018-01-12 Compositions et procédés pour perturber les mécanismes moléculaires associés à un dysfonctionnement mitochondrial et à une maladie neurodégénérative

Country Status (2)

Country Link
US (2) US11352625B2 (fr)
WO (1) WO2018132755A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110079551A (zh) * 2019-04-03 2019-08-02 广州辉园苑医药科技有限公司 一种环状rna表达载体及其构建方法和应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050266459A1 (en) * 2004-05-04 2005-12-01 Poulsen Tim S Nucleic acid probes and nucleic acid analog probes
WO2006083854A2 (fr) * 2005-01-31 2006-08-10 Perlegen Sciences, Inc. Base genetique de la maladie d'alzheimer et son diagnostic et son traitement
US20100183610A1 (en) * 2006-07-14 2010-07-22 Celera Corporation Genetic polymorphisms associated with alzheimer's disease, methods of detection and uses thereof
US20130164845A1 (en) * 2011-12-23 2013-06-27 Kevin Polach Compositions and Methods for the Delivery of Biologically Active RNAs
US20150051212A1 (en) * 2012-03-28 2015-02-19 Regenerative Research Foundation Compositions And Methods For Inhibiting Drusen
US20150073025A1 (en) * 2008-08-12 2015-03-12 Zinfandel Pharmaceuticals, Inc. Method for identifying disease risk factors
US20150141320A1 (en) * 2012-05-16 2015-05-21 Rana Therapeutics, Inc. Compositions and methods for modulating gene expression
WO2017214471A1 (fr) * 2016-06-10 2017-12-14 Saunders, Ann M. Procédés de détection de variants structurels dans une maladie neurodégénérative

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6077709A (en) * 1998-09-29 2000-06-20 Isis Pharmaceuticals Inc. Antisense modulation of Survivin expression

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050266459A1 (en) * 2004-05-04 2005-12-01 Poulsen Tim S Nucleic acid probes and nucleic acid analog probes
WO2006083854A2 (fr) * 2005-01-31 2006-08-10 Perlegen Sciences, Inc. Base genetique de la maladie d'alzheimer et son diagnostic et son traitement
US20100183610A1 (en) * 2006-07-14 2010-07-22 Celera Corporation Genetic polymorphisms associated with alzheimer's disease, methods of detection and uses thereof
US20150073025A1 (en) * 2008-08-12 2015-03-12 Zinfandel Pharmaceuticals, Inc. Method for identifying disease risk factors
US20130164845A1 (en) * 2011-12-23 2013-06-27 Kevin Polach Compositions and Methods for the Delivery of Biologically Active RNAs
US20150051212A1 (en) * 2012-03-28 2015-02-19 Regenerative Research Foundation Compositions And Methods For Inhibiting Drusen
US20150141320A1 (en) * 2012-05-16 2015-05-21 Rana Therapeutics, Inc. Compositions and methods for modulating gene expression
WO2017214471A1 (fr) * 2016-06-10 2017-12-14 Saunders, Ann M. Procédés de détection de variants structurels dans une maladie neurodégénérative

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DEANDRADE, A ET AL.: "Genetic and epigenetic variations contributed by Alu retrotransposition", BIOMED CENTRAL GENOMICS, vol. 12, no. 1, 20 December 2011 (2011-12-20), pages 1 - 13, XP021130451 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110079551A (zh) * 2019-04-03 2019-08-02 广州辉园苑医药科技有限公司 一种环状rna表达载体及其构建方法和应用

Also Published As

Publication number Publication date
US11932855B2 (en) 2024-03-19
US20210155925A1 (en) 2021-05-27
US11352625B2 (en) 2022-06-07
US20220251558A1 (en) 2022-08-11

Similar Documents

Publication Publication Date Title
US20210108208A1 (en) Targeted augmentation of nuclear gene output
CN105960459B (zh) 抑制neat1用于治疗实体肿瘤
AU2016335572B2 (en) Compositions and methods for treating Huntington's disease and related disorders
Henshall et al. Epigenetics and epilepsy
Østergaard et al. Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS
EP3390642B1 (fr) Compositions et procédés pour le traitement de rétinite pigmentaire 13
Volonté et al. MicroRNAs: newcomers into the ALS picture
US20100151470A1 (en) Methods and compositions for locating snp heterozygosity for allele specific diagnosis and therapy
JP2010539990A (ja) アンチセンス転写物を標的とするａｇＲＮＡおよびギャップマーを用いた遺伝子発現の調節方法
CA2726866A1 (fr) Procedes et compositions pour le traitement de la maladie de huntington
Lennox et al. Mini-review on current strategies to knockdown long non-coding RNAs
Gatto et al. Epigenetic alteration of microRNAs in DNMT3B-mutated patients of ICF syndrome
Carvill et al. Haploinsufficiency, dominant negative, and gain-of-function mechanisms in epilepsy: matching therapeutic approach to the pathophysiology
US11932855B2 (en) Compositions and methods for disrupting the molecular mechanisms associated with mitochondrial dysfunction and neurodegenerative disease
WO2004078941A2 (fr) Modulation de l'expression genetique au moyen d'hybrides adn/arn
US9023819B2 (en) Treatment of a disease or a condition associated with aberrant gene hypomethylation by a method involving tailored epigenomic modification
WO2012164058A1 (fr) Procédés pour l'ajustement de l'expression du génome mitochondrial par un microarn
EP3746555B1 (fr) Inhibition d'un lncrna pour le traitement du neuroblastome
CA2960728A1 (fr) Therapie allele-specifique pour les haplotypes de la maladie d'huntington
de Vrieze et al. Allele-specific antisense oligonucleotide therapy for dominantly inherited hearing impairment DFNA9
da Rocha Loureiro Non-coding repeat insertion and RNA-mediated neurodegeneration
WO2023018637A1 (fr) Édition génique d'éléments régulateurs

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18739171

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18739171

Country of ref document: EP

Kind code of ref document: A1